Molecular analysis of the heavy chain variable region genes of human hybridoma clones specific for coagulation factor VIII

Soheila Gharagozlou; Gholam Ali Kardar; Hodjattallah Rabbani; Fazel Shokri

doi:10.1160/TH05-06-0445

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2005; 94(06): 1131-1137
DOI: 10.1160/TH05-06-0445

Blood Coagulation, Fibrinolysis and Cellular Haemostasis

Schattauer GmbH

Molecular analysis of the heavy chain variable region genes of human hybridoma clones specific for coagulation factor VIII

Authors

Soheila Gharagozlou

¹Department of Immunology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran

²National Cell Bank of Iran, Pasteur Institute of Iran, Tehran, Iran
Gholam Ali Kardar

¹Department of Immunology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran

³Immunology, Asthma and Allergy Research Institute, Tehran University of Medical Sciences, Tehran, Iran
Hodjattallah Rabbani

⁴Monoclonal Antibody Research Center, Avesina Research Center, Tehran, Iran

⁵Immune and Gene Therapy Lab, Cancer Center Karolinska, Karolinska Hospital, Stockholm, Sweden
Fazel Shokri

¹Department of Immunology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran

²National Cell Bank of Iran, Pasteur Institute of Iran, Tehran, Iran

⁴Monoclonal Antibody Research Center, Avesina Research Center, Tehran, Iran

Abstract

Summary

Hemophilia A is a X-linked hematologic disorder characterized by undetectable or low amounts of functional coagulation factor VIII (FVIII). Replacement therapy induces FVIII neutralizing antibody (Ab) (inhibitor) in a proportion of patients which makes further treatment of these patients ineffective and costly. To envisage mechanisms underlying inhibitor development, seven hybridoma clones specific for FVIII were generated from two hemophiliaA patients with high titer of inhibitor. Specificity and isotype of the monoclonal antibodies (mAbs) were determined by ELISA. Immunoglobulin (Ig) variable region heavy (V_H) chain gene family usage was identified by RT-PCR usingV_H1–6 specific primers. Nucleotide sequences of the V_H gene of FVIII specific clones were determined and aligned to the most homologous germ line genes in the GenBank. Analysis of the expressed V_H genes by RT-PCR revealed that the hybridomas utilized either the V_H1 (71%) or the V_H3 (29%) gene family. Three V_H domains were encoded by V1–69 (DP-10),V1–2 (DP-8),andV1–8 (DP-15) genes and two by V1–18 (DP-14) gene, all from the V_H1 gene family. Of the V_H3-gene family expressing clones, one belonged to V3–66 (DP-86) and the other one toV3–21 (DP-77) germline genes. The CDR3 length was found to be highly different amongst these clones ranging from 11 to 22 amino acid residues. These data suggest that FVIII-specific Abs preferentially use V_H gene segments derived from V_H1 gene family. Diversity of the expressed V_H genes and their CDR3 length implies that different epitopes are recognized by these mAbs.

Keywords

Hemophilia A - factor VIII - human hybridoma - V_H gene

Full Text

References

References
1 Vehar GA, Keyt B, Eaton D. et al Structure of human factor VIII. Nature 1984; 312: 337-42.
2 Butenas S, Mann KG. Blood coagulation. Biochemistry 2002; 67: 3-12.
3 Hoyer LW. Why do many hemophilia A patients develop an inhibitor?. Br J Haematol 1995; 90: 498-501.
4 Kreuz W, Escuriola-Ettingshausen C, Matinez-Saguer I. et al Epidemiology of inhibitors in hemophilia A. Vox Sang 1996; 70 (Suppl. 01) 2-8.
5 Cohen AJ, Kessler CM. Acquired inhibitors. Baillieres Clin Haematol 1996; 9: 331-54.
6 Scandella D, Mattingly M, De Graaf S. et al Localization of epitopes for human factor VIII inhibitor antibodies by immunoblotting and antibody neutralization. Blood 1989; 74: 1618-26.
7 Rajewsky K. Clonal selection and learning in the antibody system. Nature 1996; 381: 751-8.
8 Van den Brink EN, Turenhout EAM, Davis J. et al Human antibodies with specificity for the C2 domain of factor VIII are derived from V_H1 germline genes. Blood 2000; 95: 558-63.
9 Van den Brink EN, Turendhout EAM, Bank CMC. et al Molecular analysis of human anti-factor VIII antibodies by V gene phage display identifies a new epitope in the acidic region following the A2 domain. Blood 2000; 95: 540-545.
10 Van den Brink EN, Turenhout EAM, AM Bovenschen. et al Multiple V_H genes are used to assemble human antibodies directed toward the A3-C1 domains of factor VIII. Blood 2001; 97: 966-72.
11 Van den Brink EN, Brill WS, Turenhout EAM. et al Two classes of germline genes both derived from the V_H1 family direct the formation of human antibodies that recognize distinct antigenic sites in the C2 domain of factor VIII. Blood 2002; 99: 2828-34.
12 Jacquemin MG, Desqueper BG, Benhida A. et al Mechanism and kinetics of factor VIII inactivation: Study with an IgG4 monoclonal antibody derived from a hemophilia A Patient with inhibitor. Blood 1998; 92: 496-506.
13 Jacquemin M, Benhida A, Peerlinck K. et al A human antibody directed to the factor VIII C1 domain inhibits factor VIII cofactor activity and binding to von Willebrand factor. Blood 2000; 95: 156-63.
14 Borrebaeck CAK. In vitro immunization for the production of murine and human monoclonal antibodies: Present status. Trends Biotechnol 1986; 4: 147-55.
15 Gharagozlou S, Ghods R, Samadi Bahrami Z. et al Characterization of human hybridoma clones isolated from hemophilia patients with specificity for different domains of coagulating factor VIII. Hum Antibodies 2003; 12: 67-76.
16 Payam khaja Pasha R, Roohi A, Shokri F. Establishment of heterohybridoma and lymphoblastoid cell lines specific for the Rh D and C antigens. Transfus Med 2003; 13: 83-92.
17 Shokri F, Mageed RA, Jefferis R. A quantitative ELISA for measurement of RF-associated CRI in serum from patients with rheumatic disease. Br J Rheum 1993; 32: 862-9.
18 Willems P, Verhagen O, Segeren C. et al Consensus strategy to quantitate malignant cells in myeloma patients is validated in a multicenter study. Belgium-Dutch Hematology-Oncology Group. Blood 2000; 96: 63-70.
19 Schettino EW, Chai SK, Kasain MT. et al VHDJH gene sequences and antigen reactivity of monoclonal antibodies produced by human B-1 cells: evidence for somatic selection. J Immunol 1997; 158: 2477-89.
20 Saenko El, Scandella D. The acidic region of the factor VIII light chain and the C2 domain together form the high affinity binding site for von Willebrand factor. J Biol Chem 1997; 272: 18007-14.
21 Sugihara T, Takahashi I, Kojima T. et al Identification of Plasma antibody epitopes and gene abnormalities in Japanese hemophilia A patients with factor VIII inhibitor. Nagoya J Med Sci 2000; 63: 25-39.
22 Muhle C, Schulz-Drost S, Khrenov AV. et al Epitope mapping of polyclonal clotting factor VIII-inhibitory antibodies using phage display. Thromb Haemost 2004; 91: 619-25.
23 Matsuda F, Ishii K, Bourvagnet P. et al The complete nucleotide sequence of the human immunoglobulin heavy chain variable region locus. J Exp Med 1998; 188: 2151-62.
24 Matsuda F, Honjo T. The human immunoglobulin V_H locus. Immunologist 1999; 7: 171-6.
25 Brezinschek HP, Foster SJ, Brezinschek Rl. et al Analysis of the human V_H gene repertoire: Differential effects of selection and somatic hypermutation on human peripheral CD5⁺/IgM⁺ and CD5⁻/IgM⁺ B cells. J Clin Invest 1997; 99: 2488-501.
26 De Wildt RMT, Tomlinson IM, Van Venrooij WJ. et al Comparable heavy and light chain pairings in normal and systemic lupus erythmatosus IgG⁺ B cells. Eur J Immunol 2000; 30: 254-61.
27 Gharagozlou S, Sharifian RA, Mageed RA. et al Analysis of expressed immunoglobulin variable region heavy chain gene products in paraproteins from Iranian patients with multiple myeloma. Path Oncol Res 2000; 6: 185-90.
28 Shokri F, Mageed RA, Richardson P. et al Jefferis R. Modulation and high frequency expression of autoantibody-associated cross-reactive idiotypes linked to the VHI subgroup in CD5-expressing B lymphocytes from patients with chronic lymphocytic leukaemia (B-CLL). Scand J Immunol 1993; 37: 673-9.
29 Shokri F, Mageed RA, Maziak BR. et al Lymphoproliferation in primary Sjogren’s syndrome. Evidence of selective expansion of a B cell subset characterized by the expression of cross-reactive idiotypes. Arthritis Rheum 1993; 36: 1128-36.
30 Shokri F, Mageed RA, Richardson P. et al Immunophenotypic and idiotypic characterization of the leukaemic B-cells from patients with prolymphocytic leukaemia: evidence for a selective expression of immunoglobulin variable region (IGV) gene products. Leuk Res 1993; 17: 669-76.
31 Moazzeni M, Mosayyebi G, Stevenson FK. et al Biased utilization of immunoglobulin variable region heavy- and light-chain genes by the malignant CD5– B lymphocytes from patients with Burkitt’s lymphoma. Int J Cancer 1994; 58: 226-32.
32 Shiokawa S, Mortari F, Lima JO. et al IgM heavy chain complementary-determining region 3 diversity is constrained by genetic and somatic mechanisms until two months after birth. J Immunol 1999; 162: 6060-70.
33 Schroeder HW, Mortari F, Shiokawa S. et al Developmental regulation of the human antibody repertoire. Ann N Y Acad Sci 1995; 764: 242-62.
34 Tonegawa S. Somatic generation of antibody diversity. Nature 1983; 302: 575-81.
35 Berek C, Milstein C. The dynamic nature of the antibody repertoire. Immunol Rev 1988; 105: 5-26.
36 Towfighi F, Gharagozlou S, Sharifian RA. et al Comparative measurement of anti-FVIII antibody by Bethesda assay and ELISA reveals restricted isotype specificity profile and epitope specificity. Acta Haematol 2005; 114: 84-90.
37 Klein U, Kuppers R, Rajewsky K. Evidence for a large compartment of IgM-expressing memory B-cells in humans. Blood 1997; 89: 1288-98.